Abstract Invariant natural killer T cells (iNKT cells) are a highly conserved lymphocyte population in mice and humans that express an invariant TCR ? chain. These lymphocytes make rapid, innate-like responses, and as a consequence, there are a number of commercial and clinical efforts to activate these cells in vivo, or expand them in vitro, in order to combat cancer, provide adjuvant or stimulating effects for vaccines, and to prevent immune-mediated diseases. It is surprising that in some cases iNKT cells stimulate Th1 or Th2 immunity and inflammation, while in other instances they are anti-inflammatory. The diversity of their effects on the immune response could be related to the selective activation of functional subsets of iNKT cells, called NKT1, NKT2 and NKT17 cells. These subsets are analogous to the well known Th1, Th2 and Th17 cells, but they differentiate in the thymus without exogenous antigenic stimulation. Our preliminary population-based and single cell RNA-Seq data show that the transcriptomes of the NKT1, NKT2 and NKT17 subsets are highly divergent, despite their similar specificity. The experiments in this application are designed to identify the differentiation steps leading to the subsets and the relationships between them, and their stability. To do this, in Aim 1 we will analyze the developmental potential of individual subsets, including iNKT cells with an intermediate or possibly transitional phenotype, using both organ cultures and by intrathymic injection. Additionally, we will use cytokine transcript fate-mapping mice to trace lineage relationships. In aims 2 and 3, we will determine what are the cell intrinsic and extrinsic factors that drive the differentiation of the iNKT cell subsets. We will analyze nonproductive ? rearrangements by next generation sequencing to provide a clonal marker, in order to determine if a single iNKT cell clone can give rise to different functional subsets. Additionally, using different types of mice in which expression of a single TCR ? is enforced, we will determine if variations in the ? chain can drive preferential subset differentiation. In aim 4, we will challenge mice with antigen, infection or chronic inflammation to determine how stable the thymus generated NKT17 subset is when confronted with inflammation or antigenic challenge. Additionally, we will analyze the transcriptomes of NKT1, NKT2 and NKT17 cells in spleen and liver, to assess the stability of the thymus-generated gene programs. Because iNKT cells do not recirculate, the transcriptome data also will provide information on the imprint of long-term residence in different organs on the iNKT cell subsets. Different populations of innate-like lymphocytes, which in addition to iNKT cells; include ?? T cells and innate lymphoid cells (ILCs), acquire during their differentiation gene programs that enforce polarized patterns of cytokine secretion. Undoubtedly there will be common themes between these cell types, and therefore our proposed work will have broader implications for understanding the differentiation of innate lymphocytes.